Dengue virus (DENV) is the most important mosquito-borne viral disease affecting humans today, with an estimated 2.5 billion living in areas at risk for epidemic transmission. Despite this, there are currently no antiviral drugs effective against dengue virus nor is there a protective vaccine. Our strategy to develop anti-DENV therapeutics is to screen libraries of small molecules of known biological activity in order to identify inhibitors and the associated cellular targets whose activity is essential for replication of the virus. Due to the centrality of their role in signal transduction and regulation of protein function, we selected kinases as a class of cellular targets that might be successfully exploited to develop effective anti-dengue therapeutics. From an initial series of screens, we found that inhibitors of Src family kinases (SFK) are potent inhibitors of DENV in vitro. Both dasatinib, a dual Src-Abl kinase inhibitor that is FDA-approved for the treatment of imatinib-resistant Chronic Myelogenous Leukemia, and AZD0530, a dual Src-Abl inhibitor that is in phase II clinical trials for the treatment of solid tumors, cause a greater than 100-fold reduction in viral titers at single-digit micromolar concentrations. SFK inhibitors are, moreover, active against dengue viral isolates representative of each of the four dengue serotypes but not against poliovirus, an unrelated RNA virus. RNAi knockdown of c-Src phenocopies the inhibitory effect of the SFK inhibitors. Collectively, our results suggest that the viral process targeted by these Src inhibitors is highly conserved and that most DENV isolates should be susceptible to the anti-viral effects of these compounds. In addition, drug- resistance should be less likely to arise since these compounds target a cellular factor that is essential for the virus but non-essential for the host. We now propose to validate SFKs as anti-DENV targets using a combination of in vitro and in vivo experiments. First, we will perform in vitro experiments to quantitatively and qualitatively characterize the effects of SFK inhibitors (including AZD0530 and dasatinib) against multiple DENV strains and in multiple cells lines. Second, in order to understand the role of SFK signal transduction in DENV biology, in Specific Aim 2 we will perform phosphoproteomic profiling experiments aimed at identifying changes in protein phosphorylation that occur upon infection of cells with dengue virus as well as which of these changes is dependent upon Src kinase activity. Using the data accumulated in vitro, we will perform experiments to evaluate the efficacy of Src kinase inhibitors in reducing viral titers and ameliorating dengue-hemorrhagic fever-like disease in a murine model of DENV infection and pathogenesis. PUBLIC HEALTH RELEVANCE Although dengue virus is the most important mosquito-borne viral disease affecting humans today, there are currently no antiviral drugs effective against dengue virus, nor is there a protective vaccine. We would like to identify small, drug-like molecules that can inhibit the virus by interacting with targets in the host cell. These small molecules can then be used to study how dengue virus interacts with its host and to develop therapies for the treatment of dengue virus infections.